6 replies to this topic

[fabipp]

Hi all!

Crystal violet can bind DNA and can also differenciate adherent cell vs non adherent cell. What the link between adherence and binding DNA???Thanks a lot!

Edited by fabipp, 14 August 2012 - 12:20 AM.

[qforthemass]

The crystal violet will bind equally well to DNA from adherent and non-adherent cells. It cannot differentiate between them per se. But through the staining protocol (e.g. the fact that one has to wash the crystal violet out at the end step) one washes away automatically the non-adherent cells.

[fabipp]

I am using crystal violet to examine biofilm formation. CV will bind to negatively charged surface molecules in the biofilm. But I think this assay that CV binds to DNA is just in mammalian cells. How it is the principle of the CV assay? Binds to DNA because the membrane is damaged, so CV will bind just to damaged cells? it is like propidium iodide? I am a bit confuse... And thanks!

[bob1]

Crystal violet primarily binds to sugar type molecules such as DNA (ribose is a sugar) and peptidoglycans (in bacteria a component of the cell wall, esp in gram+ bacteria) so can be useful for staining bacteria (it is the stain that differentiates gram+ bacteria) so long as they have enough peptidoglycan in the cell wall.
For eukaryotic cells, the cells are first fixed in something like methanol or glutaraldehyde that wil de-fat the membranes and cross-link the proteins, thus allowing the stain to permeate.

[fabipp]

Thank you very much bob 1! But so why CV it is used in Vitality test? I read that he is used to examine cell vitality. Like MTT. But what is the action of CV? Because MTT is to examine mitochondrial activity. And CV?

[bob1]

Crystal violet won't penetrate living cells I guess. You can make non-fixing solutions of it, which could be used as a viability test, but you might be better off with something like neutral red or trypan blue (they work for eukaryotic cells, but I can't vouch for bacteria)

[Zagami Francesco]

Viability cells in microplate using crystal violet
Cell culture 12 wells plates containing colonies, had to be gently washed with PBS and fixed with 3.7% formaldehyde for 10 minutes. Wells are rinsed once again with PBS and colonies are stained with 0.2% crystal violet solution in 10% ethanol for 10 minutes. Excess stain is removed by washing repeatedly with PBS. All the procedures has to be done at room temperature. The plates can be stored at room temperature or at +4 °C for several months without any visible fading of the dye. The crystal violet staining of cells from each well is solubilized using 1 ml of 10% acetic acid and the Abs of the solution is  measured at a wavelength of 590 nm.